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"Chimeric coronavirus-likeGENE_OR_GENE_PRODUCT particles carrying severe acute respiratory syndrome coronavirusORGANISM (SCoV) S protein protect miceORGANISM against challenge with SCoVSIMPLE_CHEMICAL",

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"We tested the efficacy of coronavirus-like particlesGENE_OR_GENE_PRODUCT (VLPsORGANISM) for protecting miceORGANISM against severe acute respiratory syndrome coronavirusORGANISM (SCoVSIMPLE_CHEMICAL) infection.

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Coexpression of SCoV SGENE_OR_GENE_PRODUCT protein and E, M and N proteins of mouse hepatitis virusORGANISM in 293T or CHO cellsCELL resulted in the efficient production of chimeric VLPsCELL carrying SCoV SGENE_OR_GENE_PRODUCT protein.

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Balb/c miceORGANISM inoculated with a mixture of chimeric VLPsCELL and alumSIMPLE_CHEMICAL twice at an interval of four weeks were protected from SCoVSIMPLE_CHEMICAL challenge, as indicated by the absence of infectious virus in the lungsORGAN.

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The same groups of miceORGANISM had high levels of SCoV-specific neutralizing antibodies, while miceORGANISM in the negative control groups, which were not immunized with chimeric VLPs, failed to manifest neutralizing antibodies, suggesting that SCoV-specific neutralizing antibodies are important for the suppression of viral replication within the lungsORGAN.

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Despite some differences in the cellularCELL composition of inflammatory infiltrates, we did not observe any overt lungORGAN pathology in the chimeric-VLPtreated miceORGANISM, when compared to the negative control miceORGANISM.

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Our results show that chimeric VLPCELL can be an effective vaccine strategy against SCoVSIMPLE_CHEMICAL infection.

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"Severe acute respiratory syndrome (SARS) is a newly emerged disease caused by SARS coronavirusORGANISM (SCoVSIMPLE_CHEMICAL).

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SARS originated in Southern China in 2002 and spread to five different continents causing >8000 infection and >700 deaths before its apparent eradication as a humanORGANISM infection in 2004 .

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It is not known if the virus will be reintroduced into the humanORGANISM population but ancestral coronavirusesORGANISM are widely distributed in batsORGANISM_SUBSTANCE and are thought to have adapted to civetsORGANISM and then to humansORGANISM in recent time periods [2, 3] .

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Because emerging viruses tend to reemerge as conditions change , it is highly desirable to develop safe and efficacious vaccines and/or antivirals to prevent SCoVSIMPLE_CHEMICAL infections.

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"All coronavirusesORGANISM, including SCoVSIMPLE_CHEMICAL, carry four structural proteins: nucleocapsidGENE_OR_GENE_PRODUCT (N) protein and three envelope proteins, namely spike (S) protein, a type I transmembrane glycoproteinGENE_OR_GENE_PRODUCT; envelope (E) protein; and membrane (M) proteinGENE_OR_GENE_PRODUCT, which has three membrane-spanning domains.

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Coronavirus SORGANISM protein is responsible for virus adsorption to susceptible cellsCELL through a specific virus-receptor interaction and induces membraneCELLULAR_COMPONENT fusion between viral envelope and host cell membrane .CELL

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S protein is a main player for determining coronavirus tissueORGANISM tropism, host specificity and viral pathogenicity .

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Because most coronavirusORGANISM neutralizing antibodies recognize S protein [1, 13] , it is not surprising that most of the current SCoV vaccineGENE_OR_GENE_PRODUCT candidates are either the S protein subunit itself or those carrying S protein .

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Furthermore, prophylactic administration of monoclonal antibodies directed at the SCoV SGENE_OR_GENE_PRODUCT protein protects animals against subsequent SCoVSIMPLE_CHEMICAL challenge .

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These studies point out that neutralizing antibodies that recognize SCoV SGENE_OR_GENE_PRODUCT protein are sufficient to prevent or decrease the morbidity and mortality associated with SCoVSIMPLE_CHEMICAL infection by primarily suppressing replication of the challenge virus.

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"Coronavirus-like particlesGENE_OR_GENE_PRODUCT (VLPsORGANISM) are produced from the cellsCELL coexpressing the S, M, and E proteins ; expression of the latter two proteins are sufficient for VLPCELL production .

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M protein plays a central role in virus assembly, while S protein is assembled into coronavirus particles throughORGANISM S protein-M protein interaction .

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Further, interactions of the MGENE_OR_GENE_PRODUCT protein with the RNA packaging signal of the viral RNA and with N protein drive incorporation of the helical nucleocapsid complex, which consists of the viral genome and N protein, into virus particlesORGANISM.

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Vaccinia virusORGANISM and/or alphavirusORGANISM replicons have been used to express coronavirusORGANISM proteins to enable generation of VLPsCELL , while we have reported production of SCoV VLPsORGANISM from 293T cellsCELL that are co-transfected with four eukaryotic pCAGGS-based expression plasmids, each of which encodes SCoV SGENE_OR_GENE_PRODUCT, MGENE_OR_GENE_PRODUCT, N and E proteins .

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Others have also reported production of SCoV VLPGENE_OR_GENE_PRODUCT from insect cellsCELL [37, 38] and mammalian cellsCELL .

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"During our studies of coronavirusORGANISM assembly, we found an efficient production of chimeric VLPsCELL carrying SCoV SGENE_OR_GENE_PRODUCT protein and murine coronavirusORGANISM (mouse hepatitis virusORGANISM or MHV) M, N and E proteins from cellsCELL coexpressing those proteins.

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In miceORGANISM immunized with the chimeric VLPsCELL, the present study describes elicitation of antibodies that neutralized SCoVSIMPLE_CHEMICAL and suppressed challenged SCoVSIMPLE_CHEMICAL replication in the lungsORGAN.

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These findings suggest that the use of chimeric VLPCELL is an effective vaccine strategy against SCoVSIMPLE_CHEMICAL infection.

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"Vero E6 cellsCELL, 293T cellsCELL and CHO cellsCELL were grown in Dulbecco'sORGANISM modified minimum essential medium (DMEMSIMPLE_CHEMICAL) supplemented with penicillinSIMPLE_CHEMICAL (100 units/ml), streptomycinSIMPLE_CHEMICAL (100 g/mlSIMPLE_CHEMICAL), 0.2% sodium bicarbonateSIMPLE_CHEMICAL and 10% fetal bovine serum (FBSORGANISM_SUBSTANCE).

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The Urbani strain of SCoVSIMPLE_CHEMICAL was obtained from T.G. Ksiazek at the Centers for Disease Control and Prevention (Atlanta, GAGENE_OR_GENE_PRODUCT), and a working stockORGANISM of this virus was prepared by serially passaging a portion of the seed virus twice in Vero E6 cellsCELL.

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The culture fluid from infected cellsCELL was clarified by low-speed centrifugation and was filtered using a 0.45 m filter, portioned, and frozen at \u221280 \u2022 C. Medium from uninfected Vero E6 cellsCELL was similarly treated and processed.

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"The plasmids, pCAGGS-MHV-A59/SGENE_OR_GENE_PRODUCT, pCAGGS-MHV-A59/E, pCAGGS-MHV-A59/MCELL, and pCAGGS-A59/NSIMPLE_CHEMICAL, were constructed by inserting the entire region of MHVORGANISM structural proteins, S, E, M and N, respectively, into a chicken beta-actinORGANISM promoterbased expression plasmid, pCAGGS-MCS; plasmid pMH54 was obtained from Paul Masters and used as a template for PCR amplification of MHVORGANISM structural genes.

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Construction of plasmids, pCAGGS-SSIMPLE_CHEMICAL expressing SCoV SGENE_OR_GENE_PRODUCT protein, pCAGGS-EGENE_OR_GENE_PRODUCT expressing SCoV EGENE_OR_GENE_PRODUCT protein, pCAGGS-MGENE_OR_GENE_PRODUCT expressing SCoV MGENE_OR_GENE_PRODUCT protein, and pCAGGS-N expressing SCoV NGENE_OR_GENE_PRODUCT protein, was described previously .

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"For preparation of MHV VLPsORGANISM, subconfluent 293T cell culturesCELL in 100-mm tissueTISSUE culture dishes were cotransfected with 14 g of pCAGGS-MHV-A59/SGENE_OR_GENE_PRODUCT, 0.4 g of pCAGGS-MHV-A59/E, 3.5 g of pCAGGS-MHV-A59/MCELL, and 14 g of pCAGGS-A59/N using TransIT-293GENE_OR_GENE_PRODUCT reagent (Mirus).

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At 3 days posttransfection, the culture media were collected, centrifuged at 1550 \u00d7 g for 10 min and filtered through a 0.45-m filter to remove cellCELL debris.

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The MHV VLPsORGANISM were pelleted down through 20% sucroseSIMPLE_CHEMICAL cushion at 26,000 rpm for 3 h by using a Beckman SW 28 rotor.

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After the pellets were suspended in NTE bufferSIMPLE_CHEMICAL (100 mM NaCl, 10 mM Tris-HCl, pH 7.0, 1 mM EDTA), the VLPsORGANISM_SUBSTANCE were centrifuged using a Beckman SW 28 rotor at 26,000 rpm for 3 h on a discontinuous sucroseSIMPLE_CHEMICAL gradient consisting of 60, 50, 30, and 20% sucroseSIMPLE_CHEMICAL.

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The VLPsCELL at the interface of 30 and 50% sucroseSIMPLE_CHEMICAL were collected, diluted and further purified on a discontinuous sucroseSIMPLE_CHEMICAL gradient consisting of 60, 50, 30, and 20% sucroseSIMPLE_CHEMICAL at 26,000 rpm for 18 h. Purified and concentrated VLPsCELL at the interface between 50 and 30% sucroseSIMPLE_CHEMICAL were collected, diluted and pelleted through a 20% sucroseSIMPLE_CHEMICAL cushion at 26,000 rpm for 2 h. The pellets were suspended in NTE bufferSIMPLE_CHEMICAL and kept at \u221280 \u2022 CGENE_OR_GENE_PRODUCT until further use.

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For preparation of SCoV VLPCELL, a mixture of 2.8 g of pCAGGS-S, 13.2 g of pCAGGS-EGENE_OR_GENE_PRODUCT, 1.4 g of pCAGGS-MGENE_OR_GENE_PRODUCT, and 2.8 g of pCAGGS-N was cotransfected into 293T cellsCELL grown on a 100-mm tissueTISSUE culture plate.

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Chimeric VLPsORGANISM were generated by transfecting 293T cellsCELL or CHO cellsCELL in a 100-mm tissueTISSUE culture plate with 14 g of pCAGGS-S, 0.4 g of pCAGGS-MHV-A59/E, 3.5 g of pCAGGS-MHV-A59/MCELL, and 14 g of pCAGGS-A59/N. SCoV VLPs and chimeric VLPs were purified using the same procedure as described for the MHV VLPORGANISM purification.

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"The total protein concentration of VLPsORGANISM was quantitated by Bio-Rad DC protein assay according to the manufacturer's instructions (BIO-RAD, CA).

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For detection of MHV S,ORGANISM MGENE_OR_GENE_PRODUCT and N proteins, we used anti-MHV serum, which was produced by immunizing rabbitsORGANISM with a purified JHMORGANISM strain of MHVORGANISM, kindly provided by Susan Baker, Loyola University of Chicago.

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For detection of SCoV SGENE_OR_GENE_PRODUCT protein, a mixture of purified rabbit polyclonal anti-SCoV S protein antibodyORGANISM (ABGENT, catORGANISM.

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no. AP6000aORGANISM) and polyclonal rabbit anti-SCoV S protein antibodyORGANISM (IMGENEXGENE_OR_GENE_PRODUCT, catORGANISM.

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SCoV NSIMPLE_CHEMICAL protein was detected by using rabbit polyclonal anti-SCoV N protein antibodyORGANISM (IMGENEXGENE_OR_GENE_PRODUCT, catORGANISM.

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no. IMG 548), and SCoV MGENE_OR_GENE_PRODUCT protein was detected by using a mixture of SCoV PUPMGENE_OR_GENE_PRODUCT antibody-N-terminal (ABGENT, catORGANISM.

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no. AP6008aORGANISM), SCoV PUPM antibodyGENE_OR_GENE_PRODUCT C-terminal (ABGENT, catORGANISM.

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no. AP6008b) and anti-SCoV M antibodiesGENE_OR_GENE_PRODUCT (ProSciGENE_OR_GENE_PRODUCT, catORGANISM.

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no. 3527P and cat no. 3529PORGANISM).

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"After washing the gel with water, proteins in the gel were stained by soaking the gel with Bio-Safe Coomassie Stain (BIO-RAD)SIMPLE_CHEMICAL with gentle agitation for 1 h. The gel was rinsed extensively with water overnight.

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"Carbon-coated, 200-mesh copper grids were floated on drops of chimeric VLPORGANISM_SUBSTANCE for 10 min.

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After washing the grids with water three times, negative staining was performed using 2% phosphotungstic acidSIMPLE_CHEMICAL (pH 7.0) for 1 min.

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"section": "ElectronCELLULAR_COMPONENT microscopic analysis of chimeric VLPCELL"

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"Six-to 8-week-oldORGANISM, female Balb/c miceORGANISM (Charles River laboratory, Wilmington, MAGENE_OR_GENE_PRODUCT) were housed in cages covered with barrier filters in an approval biosafety level 3 animal facility maintained by the University of Texas Medical Branch at Galveston, Texas.

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All of the mouseORGANISM experiments were performed using experimental protocols approved by the University of Texas Medical Branch Investigational Animal Care and Use Committee; all of the experiments were carried out following National Institutes of Health and United States Department of Agriculture guidelines.

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"MHV VLPsORGANISM, chimeric VLPsORGANISM or influenza virus vaccineORGANISM were adjusted to twice their desired final concentration (g/0.1 ml) and then mixed with an equal volume of phosphate buffered salineSIMPLE_CHEMICAL (PBS) or alumSIMPLE_CHEMICAL adjuvant (Imject alum, PierceGENE_OR_GENE_PRODUCT, catORGANISM.

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At day 0, they were administered intramuscularly (i.m.) using 1-ml tuberculin syringes (Norm-JectGENE_OR_GENE_PRODUCT, Tuttingen, Germany) and 26-g, 3/8-in.

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beveled needles (Becton Dickinson, catORGANISM.

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In control groups, miceORGANISM were inoculated i.m.

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with Vero E6 cellCELL culture fluid, a mixture of influenza vaccine and alumSIMPLE_CHEMICAL, or alumSIMPLE_CHEMICAL alone, or they were leftORGAN untreated.

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MiceORGANISM were inoculated on day 0 and, as indicated in the bodyORGANISM_SUBDIVISION of the paper a second injection was given on day 28 in most experiments.

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SCoVSIMPLE_CHEMICAL was inoculated directly into the naresORGAN of miceORGANISM (40 l) that were lightly anesthetized with isofluraneSIMPLE_CHEMICAL (IsoFlo; Abbott Laboratories; North Chicago, ILGENE_OR_GENE_PRODUCT).

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MiceORGANISM that were inoculated with 1 \u00d7 10 6 TCID 50 of SCoVSIMPLE_CHEMICAL at day 0 to provide post-infection immunity did not undergo a second inoculation.

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In most of our experiments, immunized miceORGANISM were challenged by intranasal (i.n.) inoculation of 1 \u00d7 10 6 TCID 50 of SCoVSIMPLE_CHEMICAL at day 56 and they were euthanized at day 58, while in some experiments miceORGANISM were inoculated with SCoVSIMPLE_CHEMICAL at day 28 and they were euthanized at day 30.

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"Procedures for immunization and SCoVSIMPLE_CHEMICAL challenge"

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"MiceORGANISM were anesthetized with isofluraneSIMPLE_CHEMICAL and then bled from the retro-orbital sinusMULTI-TISSUE_STRUCTURE plexus.

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After heat inactivation at 56 \u2022 C for 30 min, seraORGANISM_SUBSTANCE were stored at 4 \u2022 C. The assay for virusspecific neutralizing antibodies was performed on serial 2-fold diluted samples of each serumORGANISM_SUBSTANCE beginning at a dilution of 1:5 using 2% FBS-DMEMORGANISM_SUBSTANCE as the diluent in 96-well tissueTISSUE culture plates (Falcon 3072); the final volume of the serially diluted samples in each well was 60 l.CELL After addition of 120 TCID 50 of SCoVSIMPLE_CHEMICAL in 60 l into each well, the samples were incubated for 45-60 min at room temperature.

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Then 100 l of these mixtures, containing 100 TCID 50 of SCoVSIMPLE_CHEMICAL, were transferred into duplicate wells of confluent Vero E6 cellsCELL grown in 96-well microtiter plates.

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After 72 h incubation, when the virus control wells exhibited advanced virus-induced CPE, the neutralizing capacity of individual serum samplesORGANISM_SUBSTANCE was assessed by determining the presence or absence of virusinduced CPECELL.

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SCoV-specific neutralizing antibody titers were expressed as the reciprocal of the last dilution of serumORGANISM_SUBSTANCE that completely inhibited virus-induced CPE.

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"Two days post SCoVSIMPLE_CHEMICAL challenge, miceORGANISM were euthanized and their lungsORGAN were removed.

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Lung lobesMULTI-TISSUE_STRUCTURE, including right middle lobeCANCER, right lower lobeCANCER, accessory lobeCANCER and left lobeCANCER, were removed and placed in 10% buffered formalin for subsequent histological examination and immunohistochemistry (IHC) as described previously .

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For virus titration, the remaining lobesORGAN of the lungsORGAN were weighed and frozen at \u221280 \u2022 C before being homogenized in PBS/10% FBSORGANISM_SUBSTANCE solution using the TissueLyserCANCER (Qiagen; Retsch, Haan, Germany).

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The homogenatesORGANISM were then centrifuged, and SCoVSIMPLE_CHEMICAL titers in the clarified fluid were determined in a TCIDSIMPLE_CHEMICAL 50 assay in quadruplicate wells of Vero E6 cellsCELL in 96-well plates.

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Titers of virus in lung homogenatesCELL were expressed as TCIDSIMPLE_CHEMICAL 50 /gram of the lungsORGAN (log 10 ) and the minimal detectable level of virus was 2.3 log 10 TCID 50 /g.

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"section": "Collection of lungsORGAN, histology, immunohistochemistry, and virus titration"

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"Formalin-fixedORGAN and paraffin-embedded tissue sectionsTISSUE were subjected to the standard hematoxylin and eosin and IHCGENE_OR_GENE_PRODUCT staining for evaluating histopathology and detecting SCoV antigenGENE_OR_GENE_PRODUCT, respectively .

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"section": "Collection of lungsORGAN, histology, immunohistochemistry, and virus titration"

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"By using a statistical program Instat, (version 3, Graphpad Software, Inc., San Diego, CA), we performed the Kruskal-Wallis non-parametric analysis of variance (ANOVA) test to compare arithmetic mean SCoV lungORGAN titers and neutralizing antibody titers; undetectable virus titers were assigned a value of 1.8 (the minimal detection limit being 2.3 log 10 /g lungORGAN) and undetectable virus-specificGENE_OR_GENE_PRODUCT neutralizing titers were assigned a value of 10.

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"We have previously reported the successful production of SCoV VLPsCELL from 293T cellsCELL cotransfected with four pCAGGSbasedGENE_OR_GENE_PRODUCT plasmids, each of which encodes SCoV SGENE_OR_GENE_PRODUCT, MGENE_OR_GENE_PRODUCT, N or E proteins without using exogenous viruses for protein expression.

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In the study reported here we varied the amount of each plasmid for cotransfection and were able to generate approximately 1.3 g of SCoV VLPsORGANISM from 2 \u00d7 10 7 293T cellsSIMPLE_CHEMICAL.

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Colloidal Coomassie blueGENE_OR_GENE_PRODUCT staining and Western blot analysis identified SCoV SGENE_OR_GENE_PRODUCT, N and MGENE_OR_GENE_PRODUCT proteins in the purified SCoV VLPsCELL (Fig. 1ACELL) .

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Likewise, transfection of four pCAGGSbased plasmidsGENE_OR_GENE_PRODUCT, each of which encoded MHV S,ORGANISM E, M or N proteins, resulted in production of MHV VLPsORGANISM (Fig. 1A) .

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After optimization, we obtained approximately 22.3 g of purified MHV VLPsORGANISM from 2 \u00d7 10 7 293T cells, demonstrating that the efficiency of MHV VLPORGANISM production was substantially better than that of SCoV VLPORGANISM production.

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Because M and E proteins drive VLPCELL assembly and release , and coronavirus S proteinORGANISM is incorporated into VLPsCELL through M-S interaction [25, 27, 28, 42] , these data indicated that MHV MORGANISM and EGENE_OR_GENE_PRODUCT proteins resulted in more efficient VLPORGANISM_SUBSTANCE production than did SCoV MGENE_OR_GENE_PRODUCT and EGENE_OR_GENE_PRODUCT proteins.

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"Production and characterization of chimeric VLPsCELL"

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"We next tested whether coexpression of SCoV SGENE_OR_GENE_PRODUCT protein and MHV EORGANISM, N and MGENE_OR_GENE_PRODUCT protein could result in the efficient production of chimeric VLPsORGANISM_SUBSTANCE which would contain high concentrations of the SCoV SGENE_OR_GENE_PRODUCT protein.

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If SCoV SGENE_OR_GENE_PRODUCT protein interacts with MHV MORGANISM protein, then the robust VLPCELL production machinery driven by MHV MORGANISM and EGENE_OR_GENE_PRODUCT proteins, could result in efficient chimeric VLPCELL production.

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Chimeric VLPCELL production, indeed, occurred from 293T cellsCELL that were cotransfected with plasmids, each of which expressed SCoV SGENE_OR_GENE_PRODUCT protein, MHV N proteinORGANISM, MHV M protein or MHV E protein (Fig. 1A) .

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Colloidal Coomassie blueGENE_OR_GENE_PRODUCT staining and Western blot analysis of purified chimeric VLPCELL showed the presence of SCoV SGENE_OR_GENE_PRODUCT, MHV NORGANISM and MHV M proteins in chimeric VLPsCELL, with only a low level of host protein contamination.

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Negative staining of chimeric VLPCELL revealed a typical spherical coronavirusORGANISM structure with peplomers (Fig. 1B) .

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After optimization, approximately 8.7 g of purified chimeric VLPsCELL was produced from 2 \u00d7 10 7 293T cells; the efficiency of chimeric VLPCELL production was about 8 times better than that of SCoV VLPsORGANISM.

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"Production and characterization of chimeric VLPsCELL"

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"We next tested efficiencies of chimeric VLPCELL production from VeroCELL and CHO cellsCELL, both of which have been used for the preparation of human vaccinesORGANISM .

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By using various DNACELLULAR_COMPONENT transfection reagents and combinations of different concentrations of each plasmid for transfection, the efficiencies of chimeric VLPCELL production from Vero cellsCELL were about 10 times lower than those from 293T cellsCELL (data not shown).

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We found that when we used TransIT-293GENE_OR_GENE_PRODUCT, the production of chimeric VLPsCELL from CHO cellsCELL was about one-third of those from 293T cellsCELL in repeated experiments (data not shown).

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Prolonged incubation of transfected 293T cellsCELL and CHO cellsCELL for more than 4 days posttransfection did not improve the accumulation of chimeric VLPsCELL in the supernatant (data not shown).

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"Production and characterization of chimeric VLPsCELL"

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"For assessing the usefulness of chimeric VLPsCELL as a SCoV vaccineSIMPLE_CHEMICAL candidate, we first tested the effects of immunization of miceORGANISM with chimeric VLPs on serumORGANISM_SUBSTANCE neutralizing antibody production and inhibition of SCoVSIMPLE_CHEMICAL replication in the lungORGAN.

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Sixto 8-week-oldORGANISM female Balb/c miceORGANISM were inoculated i.m.

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once with a mixture of 2 g of chimeric VLPsCELL prepared from 293TORGANISM with placebo (medium from Vero E6 cellsCELL), a mixture of 2 g chimeric VLPCELL and PBS or a mixture of 2 g chimeric VLPCELL and alumSIMPLE_CHEMICAL or i.nORGANISM_SUBSTANCE.

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with 1 \u00d7 10 6 TCID 50 of infectious SCoV.GENE_OR_GENE_PRODUCT After 28 days, these miceORGANISM were challenged with 1 \u00d7 10 6 TCID 50 of SCoV.GENE_OR_GENE_PRODUCT Two days later, miceORGANISM were sacrificed and the virus titers in the lungsORGAN were determined as shown in the graph.

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cellsCELL and PBS or 2 g of chimeric VLPCELL mixed with alumSIMPLE_CHEMICAL.

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As a placebo, miceORGANISM were inoculated i.m.

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with Vero E6 cellCELL culture fluid, while another group of miceORGANISM were inoculated i.n.

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with 1 \u00d7 10 6 TCID 50 of SCoVSIMPLE_CHEMICAL to produce solidCANCER post-infection immunity.

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Twenty-eight days later, the miceORGANISM were bled for determining the serumORGANISM_SUBSTANCE neutralizing antibody titers ( Fig. 2A) .

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Subsequently, the miceORGANISM were challenged i.nORGANISM.

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with 1 \u00d7 10 6 TCID 50 of SCoVSIMPLE_CHEMICAL and the lung virusMULTI-TISSUE_STRUCTURE titers at 2 days post SCoVSIMPLE_CHEMICAL challenge were determined (Fig. 2B) .

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No SCoV-specific neutralizing antibodies were detectable in the seraORGANISM_SUBSTANCE of the miceORGANISM inoculated i.m.

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Minimal or no virus-specific neutralizing antibodiesGENE_OR_GENE_PRODUCT were evident in miceORGANISM inoculated with chimeric VLPsORGANISM mixed with PBS (mean titer 10 \u00b1 0).

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The mean serumORGANISM_SUBSTANCE neutralizing titers for the groups of miceORGANISM inoculated with live virus (70 \u00b1 20) or chimeric VLPCELL administered with alumSIMPLE_CHEMICAL (60 \u00b1 23) were not statistically different from each other, but were statistically significantly higher (p < 0.05) than those of the miceORGANISM immunized with a mixture of chimeric VLPsCELL and PBS and the placebo group.

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After challenge, the miceORGANISM immunized with Vero E6 cell cultureORGANISM fluid (placebo) or with a mixture of 2 g chimeric VLPCELL and PBS had high mean virus titers of 6.5 log 10 /lung and 5.2 log 10 /lung, respectively.

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In contrast, no virus was detectable in the lungsORGAN of the miceORGANISM previously administered either live SCoVSIMPLE_CHEMICAL or 2 g of chimeric VLPCELL mixed with alumSIMPLE_CHEMICAL.

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The SCoVSIMPLE_CHEMICAL titers in the lungsORGAN of these miceORGANISM two days post virus challenge were inversely proportional to their serumORGANISM_SUBSTANCE virus-neutralizing antibody titers.

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"section": "SerumORGANISM_SUBSTANCE neutralizing antibody titers and lung SCoVMULTI-TISSUE_STRUCTURE titers in the miceORGANISM immunized once with chimeric VLPsCELL"

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"To study the immunogenicity of chimeric VLPsCELL, three groups of miceORGANISM were inoculated i.m.

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with 0.5, 1, or 2 g mixed with alumSIMPLE_CHEMICAL.

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Other groups of miceORGANISM received a mixture of 2 g of chimeric VLPsCELL and PBS, 2 g of MHV VLPsORGANISM and alumSIMPLE_CHEMICAL, 1 g of Influenza A virus vaccineORGANISM and alumSIMPLE_CHEMICAL, Vero E6 cellCELL culture fluid, live SCoVSIMPLE_CHEMICAL, or alumSIMPLE_CHEMICAL alone, or the miceORGANISM received no treatment.

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Mean neutralizing titers at 28 days post inoculation in miceORGANISM receiving 0.5, 1, or 2 g chimeric VLPsCELL with alumSIMPLE_CHEMICAL were 20 \u00b1 10, 84 \u00b1 75, and 110 \u00b1 60, respectively (Fig. 3A) .

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In the absence of alumSIMPLE_CHEMICAL, 2 g chimeric VLPsCELL induced a lower but significant neutralizing antibody response of 12.5 \u00b1 5.

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The miceORGANISM infected with SCoVSIMPLE_CHEMICAL had the neutralizing titer of 70 \u00b1 20.

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The miceORGANISM inoculated with 2 g of MHV VLPORGANISM had no detectable virus-specific neutralizing antibodiesGENE_OR_GENE_PRODUCT, demonstrating that MHV SGENE_OR_GENE_PRODUCT protein did not elicit neutralizing antibodies against SCoV.GENE_OR_GENE_PRODUCT The neutralizing antibody titers at 28 days post inoculation were not investigated in the groups of miceORGANISM inoculated with Influenza vaccine and alumSIMPLE_CHEMICAL, alumSIMPLE_CHEMICAL alone, or in those receiving no treatment.

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"Effects of chimeric VLPCELL amounts on neutralizing antibody titers and inhibition of challenged SCoVSIMPLE_CHEMICAL replication in the lungsORGAN"

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At day 28, these miceORGANISM, except for those untreated and those that had been inoculated with live SCoVSIMPLE_CHEMICAL, were boosted with the same material.

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MiceORGANISM injected with chimeric VLPs and alumSIMPLE_CHEMICAL had no increase in titer at the lowest dose, but 1-and 2-g doses resulted in marked booster responses.

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Animals receiving the highest dose developed titers of 200 \u00b1SIMPLE_CHEMICAL 97.7, while those infected with SCoVSIMPLE_CHEMICAL had the neutralizing titer of 97 \u00b1 60.

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A second inoculation of a mixture of 2 g chimeric VLPs with PBS efficiently boosted median neutralizing antibody titers to 57 \u00b1 21, which was higher than the titer following 0.5 g of chimeric VLPCELL mixed with alumSIMPLE_CHEMICAL (15 \u00b1 5.7).

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No neutralizing antibodies were detected at the lowest dilution tested in groups receiving MHV VLPORGANISM, influenza vaccine and alumSIMPLE_CHEMICAL, alumSIMPLE_CHEMICAL alone, or placebo or in those leftORGAN untreated.

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"Effects of chimeric VLPCELL amounts on neutralizing antibody titers and inhibition of challenged SCoVSIMPLE_CHEMICAL replication in the lungsORGAN"

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"All miceORGANISM were challenged i.n.

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with live SCoVSIMPLE_CHEMICAL on day 56 and sacrificed 2 days later.

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As expected, maximal pulmonary virusCELL titers were detected in the groups of miceORGANISM inoculated with placebo (6.9 log 10 \u00b10.7), MHV VLPsORGANISM (6.2 log 10 \u00b10.4GENE_OR_GENE_PRODUCT), influenza vaccine and alumSIMPLE_CHEMICAL (7.5 \u00b1 0.1), alumSIMPLE_CHEMICAL alone (7.3 \u00b1 0), and in those leftORGAN untreated (8.5 \u00b1 0) (Fig. 3C) .

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Partial protection was obtained in groups of miceORGANISM inoculated twice with 0.5 g chimeric VLPCELL mixed with alumSIMPLE_CHEMICAL (5.5 log 10 \u00b10.6GENE_OR_GENE_PRODUCT), 2 g chimeric VLPsCELL and PBS (3.9 log 10 \u00b11.1) or 1 g chimeric VLPsORGANISM mixed with alumSIMPLE_CHEMICAL (3.1 log 10 \u00b10.5GENE_OR_GENE_PRODUCT).

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Notably, no virus SCoV-specific neutralizing serumORGANISM_SUBSTANCE antibody titers in the immunized miceORGANISM and SCoVSIMPLE_CHEMICAL titers in lungsORGAN of mice 2ORGANISM days after intranasal challenge with SCoVSIMPLE_CHEMICAL at day 56.

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On day 0, miceORGANISM were either leftORGAN untreated or were inoculated i.nORGANISM.

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with 1 \u00d7 10 6 TCID 50 of SCoVSIMPLE_CHEMICAL or injected i.mSIMPLE_CHEMICAL.

displaCy

with placebo (medium from Vero E6 cellsCELL), a mixture of 2 g MHV VLPsORGANISM and PBS, a mixture of 2 g chimeric VLPCELL and PBS, a mixture of 2 g chimeric VLPCELL and alumSIMPLE_CHEMICAL, a mixture of 1 g chimeric VLPCELL and alumSIMPLE_CHEMICAL, a mixture of 0.5 g chimeric VLPCELL and alumSIMPLE_CHEMICAL, a mixture of 1 g influenza virus vaccine and alumSIMPLE_CHEMICAL, or alumSIMPLE_CHEMICAL alone.

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(A) The graph represents virus-specific serumORGANISM neutralizing antibody titers at 28 days post inoculation.

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(B) At 56 days blood samplesORGANISM_SUBSTANCE were collected, and then the miceORGANISM, excluding those inoculated with live SCoVSIMPLE_CHEMICAL and leftORGAN untreated, were re-inoculated with the same material.

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The graph represents virus-specificGENE_OR_GENE_PRODUCT, serumneutralizing antibody titers at 56 days after initial inoculation.

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After 2 days, miceORGANISM were euthanized, and the lung virusTISSUE titers were determined.

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The vertical dashed line in (C) denotes the minimal virus detection level in this assay (2.3 log 10 TCID 50 /g lungORGAN).

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The number of miceORGANISM used for (B) and (C) were: 7 for placebo, SCoVSIMPLE_CHEMICAL, and 2 g chimeric VLPCELL with PBS; 6 for 2 g chimeric VLPCELL with alumSIMPLE_CHEMICAL; 5 for MHV VLPORGANISM with alumSIMPLE_CHEMICAL and 1 g chimeric VLPCELL with alumSIMPLE_CHEMICAL; 4 for 0.5 g chimeric VLPCELL with alumSIMPLE_CHEMICAL; and 3 for all other groups.

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was detectable in the lungsORGAN of any of the miceORGANISM given live virus i.nORGANISM.

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or two doses of 2 g chimeric VLPCELL mixed with alumSIMPLE_CHEMICAL.

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The mean lung virus titers for the latter two groups were statistically different from the mean pulmonary SCoVMULTI-TISSUE_STRUCTURE titer seen in the placebo control group when these means were compared using a non-parametric ANOVA test (both p values being <0.01).

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Overall, the virus levels in these animals generally correlated inversely with those of SCoVspecific neutralizing antibodies present in the seraORGANISM_SUBSTANCE of the miceORGANISM.

displaCy

"Effects of chimeric VLPCELL amounts on neutralizing antibody titers and inhibition of challenged SCoVSIMPLE_CHEMICAL replication in the lungsORGAN"

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"Histopathological examination of the groups of miceORGANISM that neither produced neutralizing antibodies nor suppressed SCoVSIMPLE_CHEMICAL replication in the lungsORGAN (i.e., miceORGANISM treated with Vero E6 cellCELL culture fluid, inoculated with a mixture of influenza virus vaccine and alumSIMPLE_CHEMICAL, and with alumSIMPLE_CHEMICAL alone, as well as those leftORGAN untreated), revealed moderate interstitial pneumonia ( Fig. 4A and BCELL) .

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The bronchial epitheliumTISSUE appeared to be the main affected target with prominent cellular cytoplasmicTISSUE swelling and blebbing.

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Extensive accumulation of cellularCELL debris and necrotic epithelial cellsCELL, accompanied by inflammatory infiltrates, occurred in some bronchiolesTISSUE (Fig. 4A) .

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We also observed a moderate infiltration of mononuclear cellsCELL around peribronchiolarTISSUE and perivascular regionsTISSUE of infected tissuesTISSUE and a mild-to-moderate thickening of the bronchiolar interstitial tissuesTISSUE and alveolar wallsTISSUE with mononuclear cellCELL infiltration (Fig. 4B) .

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IHC staining demonstrated the presence of SCoV NGENE_OR_GENE_PRODUCT protein within bronchiolar epithelial cellsCELL, but not within cellsCELL of alveolar liningMULTI-TISSUE_STRUCTURE (Fig. 4C) .

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In contrast to the prominent pathology and viral replication in the lungsORGAN of the control animals, mild interstitial pneumonia was found, upon viral challenge, in the miceORGANISM previously immunized with mixtures of either chimeric VLPCELL and PBS, or chimeric VLPCELL and alumSIMPLE_CHEMICAL, and in those infected with live SCoVSIMPLE_CHEMICAL (Fig. 4D, E, G, H) .

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Specifically, cytopathologyPATHOLOGICAL_FORMATION, i.e., swelling and blebbingCELLULAR_COMPONENT, was rarely observed in bronchiolar epithelial cellsCELL, despite the presence of some desquamated cellsCELL within the airway lumenMULTI-TISSUE_STRUCTURE.

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Additionally, the thickening of bronchiolar interstitial tissuesTISSUE and the alveolar wallTISSUE and cellularCELL infiltration were less prominent when compared with those of the control groups.

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There was no big difference in the lungORGAN pathology between miceORGANISM inoculated with VLPCELL and PBS and those inoculated with VLPSIMPLE_CHEMICAL and alumSIMPLE_CHEMICAL.

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We noticed that inflammatory mononuclear cellsCELL seemed to be the main, if not only, cellularCELL component of the cellular infiltratesTISSUE around the peribronchiolarTISSUE and perivascular regionsTISSUE of miceORGANISM initially primed with live SCoV.GENE_OR_GENE_PRODUCT",

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"section": "Histopathological examination and IHC of the lungsORGAN of the immunized miceORGANISM after SCoVSIMPLE_CHEMICAL challenge"

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In addition to inflammatory mononuclear cellsCELL, we observed the infiltration of neutrophilsCELL and eosinophilsCELL around the bronchiolesTISSUE and blood vesselsMULTI-TISSUE_STRUCTURE of miceORGANISM inoculated with a mixture of chimeric VLPsCELL and PBS and that of chimeric VLPsCELL and alumSIMPLE_CHEMICAL ( Fig. 4DCANCER and E) ; counting of the infiltrating cellsCELL at the five fields of each mouseORGANISM's lung tissuesTISSUE revealed that infiltrating eosinophilsCELL represented 13.2 \u00b1 9.6% and 22.2 \u00b1 9.9% of all the infiltrating cellsCELL in miceORGANISM inoculated with a mixture of chimeric VLPCELL and PBS and in those inoculated with chimeric VLPCELL and alumSIMPLE_CHEMICAL, respectively.

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In contrast, infiltrating eosinophilsCELL represented merely 1.42 \u00b1 1.42%, 0%, 0% and 0.77 \u00b1 0.33% of all infiltrating cellsCELL in miceORGANISM inoculated, respectively, with Vero E6 cellCELL culture fluid, a mixture of influenza virus vaccine and alumSIMPLE_CHEMICAL, alumSIMPLE_CHEMICAL alone, and in those leftORGAN untreated.

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"section": "Histopathological examination and IHC of the lungsORGAN of the immunized miceORGANISM after SCoVSIMPLE_CHEMICAL challenge"

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"The present study tested the efficacy of immunization of miceORGANISM with chimeric VLPs for elicitation of anti-SCoVneutralizing antibodies, suppression of SCoVSIMPLE_CHEMICAL replication in the lungsORGAN, and SCoV-mediated lung cytopathologyCANCER.

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Although various strategies for SCoV vaccinesSIMPLE_CHEMICAL, including expression of SCoV SGENE_OR_GENE_PRODUCT protein in other viruses [18, 48, 49] , inactivated SCoV particlesSIMPLE_CHEMICAL , DNACELLULAR_COMPONENT vaccines [17, 54] , recombinant S protein [55, 56] and other approaches [57, 58] , have been reported, to our knowledge this is the first report testing VLPsCELL propagated in mammalian cellsCELL as SCoV vaccineGENE_OR_GENE_PRODUCT candidates.

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It appears that chimeric VLPsCELL produced from mammalian cellsCELL in this study were more immunogenic than were SCoV VLPsORGANISM produced by using a baculovirus-expression system ; immunization of the miceORGANISM four times with 100 g of the baculovirus-derived SCoV VLPsORGANISM in each immunization elicited neutralizing antibody titers that were no longer higher than those obtained in the present study.

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Use of inactivated SCoVSIMPLE_CHEMICAL as a SCoV vaccineSIMPLE_CHEMICAL is a similar vaccine strategy that was explored in the present study.

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Others reported that immunizing miceORGANISM twice with 1 g of double-inactivated SCoVSIMPLE_CHEMICAL with alumSIMPLE_CHEMICAL elicited high titers of neutralizing antibodies .

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A major negative aspect of the use of inactivated SCoV vaccines is that extreme care must be taken to completely inactivate infectious SCoVSIMPLE_CHEMICAL in the vaccine preparations.

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"Accumulated data suggest the importance of the compatibility of the S protein endodomain (or a cytoplasmic tailMULTI-TISSUE_STRUCTURE) and MGENE_OR_GENE_PRODUCT protein for assembly of S protein into coronavirusORGANISM or VLPsORGANISM [40, 59, 60] ; in past reports, S protein assembly occurred if these two regions are derived from the same virus, but not from different coronavirusesORGANISM.

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Accordingly, we were surprised to find the efficient production of chimeric VLPsCELL carrying SCoV SGENE_OR_GENE_PRODUCT protein and MHV-derived N, MGENE_OR_GENE_PRODUCT and E proteins.

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The present data suggest that the endodomain of SCoV SGENE_OR_GENE_PRODUCT protein interacted efficiently with MHV MGENE_OR_GENE_PRODUCT protein and assembled into VLPsCELL.

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The endodomain of coronavirus S proteinsORGANISM has two partially overlapping sub-regions, a N-terminal, \u223c18-residue-long, cysteine-rich region and a C-terminal, \u223c27-residue-longGENE_OR_GENE_PRODUCT, charge-richCELL region (Fig. 5) .

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MHVORGANISM lacking the very C-terminal 12-amino-acid segment of the S protein endodomain is viable and replicates well in cellCELL culture, suggesting that the removal of 12 carboxy-terminal residues does not inhibit interactions between the mutant S protein and the M protein [25, 26] .

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In contrast, replication is severely impaired in MHVORGANISM mutants lacking the very C-terminal 22 amino acids or 25 amino acids of the S protein endodomain, i.e., constituting the majority of the charge-richCELL region .

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Furthermore, a single point mutation of a charged amino acidAMINO_ACID to alanineAMINO_ACID in the charge-rich region of the endodomain has no detectable effect on the incorporation of a heterologous protein containing MHV S endodomain into virions, whereas mutants containing multiple charged residues-toalanineSIMPLE_CHEMICAL significantly reduce the incorporation of the protein into virions .

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These data suggest that the charge-richCELLULAR_COMPONENT region of the endodomain plays a key role in the selective inclusion of the S protein into the virion.

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We noted that there were 6 charged residues between the transmembraneCELLULAR_COMPONENT domain and the C-terminal 12 amino acids in both SCoVSIMPLE_CHEMICAL and MHVORGANISM, while only 3 charged residues are found in feline infectious peritonitis virusORGANISM (FIPVORGANISM) (Fig. 5) .

displaCy

FIPV SORGANISM protein is not assembled into VLPs or viruses that carry MHV MORGANISM, N and E proteins .

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Accordingly, the presence of the same number of charged residues at the region of the endodomain, which is considered to be important for S protein assembly into virus particles, in both SCoVSIMPLE_CHEMICAL and MHVORGANISM implies that both viruses had a similar structure in this portion of the endodomain, allowing them to interact with MHV MORGANISM protein.

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"Inoculation of miceORGANISM with a mixture of chimeric VLPsCELL and alumSIMPLE_CHEMICAL, but not that of MHV VLPsORGANISM and alumSIMPLE_CHEMICAL, induced antibodies that neutralized SCoVSIMPLE_CHEMICAL (Fig. 3) .

displaCy

The lack of heterologous protection confirmed that the SCoV SGENE_OR_GENE_PRODUCT protein is the relevant constituent of the chimeric VLPsCELL.

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As has been observed in other VLPSIMPLE_CHEMICAL systems [61, 62] , the particles themselves are immunogenic after 1 or 2 injections and provided protection when challenge was carried out after the second i.m.

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injection of only 2 g. Addition of alumSIMPLE_CHEMICAL adjuvant enhanced induction of neutralizing antibodies, and indeed titers reached those post-infection after two injections of 2 g. All VLPCELL groups had significant protection as evidenced by reduction in lungORGAN viral titers 2 days after virulent virus challenge, the optimum time to detect replication in the Balb/c mouseORGANISM .

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Higher neutralizing antibody titers correlated with lower virus content, and the 2 g plus alumSIMPLE_CHEMICAL groups had no detectable virus.

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Overall, our data were in agreement with past reports revealing the importance of SCoV-specific neutralizing antibodies, most of which recognize S protein, for curbing SCoVSIMPLE_CHEMICAL replication in the lungTISSUE [21, .

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"Immunization of miceORGANISM with Venezuelan equine encephalitis virus replicon particles encodingORGANISM SCoV NGENE_OR_GENE_PRODUCT protein induced an enhanced immunopathology, that included infiltrating eosinophilsCELL in the lungsORGAN, following SCoVSIMPLE_CHEMICAL challenge , implying the possibility that a SCoV vaccineSIMPLE_CHEMICAL lacking N protein expression may be desirable, as it would reduce the potential risk of immunopathological changes primed by immunization.

displaCy

In this regard, chimeric VLPsCELL carrying MHV N proteinORGANISM or those lacking N protein (unpublished data) could eliminate the possible induction of SCoV N protein-induced immunopathologySIMPLE_CHEMICAL.

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The efficient production of chimeric VLPCELL from CHO cellsCELL as we report in this study is promising as a new strategy for preparing VLP-based SCoV vaccine from mammalian cellsCELL; its suitability for preparing large quantities of VLP-based vaccine against SCoVSIMPLE_CHEMICAL remains to be determined.

displaCy

Further improvements of chimeric VLPCELL production, e.g., codon-optimization of the SCoV SGENE_OR_GENE_PRODUCT gene and MHV N, M andORGANISM EGENE_OR_GENE_PRODUCT genes in the expression plasmids and development of an inexpensive transfection reagent and/or procedure, would be required for the substantial enhancement of chimeric VLPCELL production.

displaCy

"Accumulated data suggest that antibodies against the FIPV SORGANISM protein fail to protect catsORGANISM from FIPVORGANISM challenge and enhance virus replication in the host through antibody-dependent enhancement (ADE) .

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It has been reported that antibodies that neutralize most human SCoV isolatesORGANISM enhance entry of a SCoVSIMPLE_CHEMICAL isolate from the civet in the cellCELL culture level , but it is unclear whether ADE occurs in animals that are immunized with SCoV vaccineSIMPLE_CHEMICAL candidates.

displaCy

We did not observe signs of ADE in our study; the miceORGANISM that were immunized with chimeric VLPs or initially inoculated with SCoVSIMPLE_CHEMICAL efficiently suppressed challenged SCoVSIMPLE_CHEMICAL replication.

displaCy

S protein in chimeric VLPsCELL was derived from a SCoV UrbaniORGANISM strain, which was also used as the challenge virus, and VLPsCELL induced antibodies that readily neutralized SCoVSIMPLE_CHEMICAL in vitro.

displaCy

In this regard, it is worth noting that immunization of miceORGANISM with a Venezuelan equine encephalitis virus replicon carrying S protein of the Urbani strainORGANISM showed only limited protection against heterologous SCoVSIMPLE_CHEMICAL that contained S gene from the human GD03ORGANISM isolate .

displaCy

"Subbarao et al. reported mild and focal peribronchiolar mononuclearCELL inflammatory infiltratesTISSUE and SCoV antigensGENE_OR_GENE_PRODUCT in bronchiolar epithelial cellsCELL in the lungsORGAN of BALB/c miceORGANISM at 2 days post SCoVSIMPLE_CHEMICAL inoculation .

displaCy

Staining with SCoV monoclonal antibodyGENE_OR_GENE_PRODUCT to N protein, we also found SCoV antigenGENE_OR_GENE_PRODUCT within bronchiolar epithelial cellsCELL after challenge of unimmunized miceORGANISM.

displaCy

Consistent with the SCoVSIMPLE_CHEMICAL titers in the lungsORGAN, no viral antigen was detected in the lungsORGAN of the miceORGANISM inoculated with chimeric VLPsCELL.

displaCy

The absence of SCoV antigenGENE_OR_GENE_PRODUCT in the lungsORGAN of VLP-immunized miceORGANISM or miceORGANISM pre-exposed to live virus, as indicated by the results of IHCGENE_OR_GENE_PRODUCT staining (Fig. 4B) , strongly argue for the efficacy of chimeric VLPsCELL in protecting against SARS-CoVORGANISM infection.

displaCy

SCoV-induced histopathological changes that were detected in unimmunized miceORGANISM in our study appeared to be similar or slightly more severe, especially with respect to the changes in the bronchiolesTISSUE, than those described in the report of Subbarao et al. .

displaCy

After SCoVSIMPLE_CHEMICAL challenge, the miceORGANISM immunized with chimeric VLPsCELL showed mixed peribronchiolar inflammatory infiltratesTISSUE and a slight thickening of the peribronchiolar interstitiumMULTI-TISSUE_STRUCTURE and alveolar wallsTISSUE.

displaCy

These lesionsPATHOLOGICAL_FORMATION in immunized miceORGANISM were milder than seen in unimmunized miceORGANISM, demonstrating the effects of chimeric VLPCELL immunization in suppressing SCoVinduced cytopathological changes in the lungsORGAN.

displaCy

The miceORGANISM immunized with a mixture of chimeric VLPCELL and alumSIMPLE_CHEMICAL had higher neutralizing antibody titers than did those immunized with a mixture of the chimeric VLPCELL and PBS, while both groups of the miceORGANISM showed similar levels of SCoV-induced lung cytopathologyCANCER, suggesting neither alumSIMPLE_CHEMICAL nor neutralizing antibody titers were sole determinant of the severity of inflammatory responses.

displaCy

Possible cellularCELL immune responses against chimeric VLPCELL proteins were suggested by the observation that the miceORGANISM inoculated with a mixture of chimeric VLPsCELL and PBS and that of chimeric VLPsCELL and alumSIMPLE_CHEMICAL, but not other groups of miceORGANISM, showed infiltration of neutrophilsCELL and eosinophilsCELL around the bronchiolesTISSUE and blood vesselsMULTI-TISSUE_STRUCTURE.

displaCy

In contrast, it has been reported that immunization of miceORGANISM with a mixture of inactivated SCoVSIMPLE_CHEMICAL with adjuvant MF59SIMPLE_CHEMICAL primary elicited humoral immune response .

displaCy

Further investigation, using not only Balb/c miceORGANISM but also other SCoVSIMPLE_CHEMICAL susceptible miceORGANISM [41, 76, 77] and other animal models [78, 79] , will be necessary to evaluate the implications and impact of cellularCELL immune activities, including pulmonaryORGAN infiltration of neutrophilsCELL and eosinophilsCELL, as well as the utility of chimeric VLPsCELL as a SCoV vaccineSIMPLE_CHEMICAL.

displaCy

"Evolutionary insights into the ecology of coronavirusesORGANISM",

displaCy

"Cross-hostCELL evolution of severe acute respiratory syndrome coronavirusORGANISM in palm civetORGANISM_SUBDIVISION and humanORGANISM",

displaCy

"The molecular biology of coronavirusesORGANISM",

displaCy

"Receptor and viral determinants of SARS-coronavirusSIMPLE_CHEMICAL adaptation to human ACE2ORGANISM",

displaCy

"Amino acidAMINO_ACID substitutions and an insertion in the spike glycoproteinGENE_OR_GENE_PRODUCT extend the host range of the murine coronavirus MHV-A59ORGANISM",

displaCy

"Murine coronavirus-induced hepatitisORGANISM: JHMORGANISM genetic background eliminates A59GENE_OR_GENE_PRODUCT spike-determined hepatotropism",

displaCy

"Recombinant avian infectious bronchitis virus expressing a heterologous spike gene demonstrates that the spike protein is a determinant of cellCELL tropism",

displaCy

"CoronavirusORGANISM spike proteins in viral entry and pathogenesis",

displaCy

"Targeted recombination demonstrates that the spike gene of transmissible gastroenteritis coronavirusORGANISM is a determinant of its enteric tropism and virulence",

displaCy

"Pathogenesis of chimeric MHV4/MHV-A59 recombinant virusesGENE_OR_GENE_PRODUCT: the murine coronavirusORGANISM spike protein is a major determinant of neurovirulenceORGANISM",

displaCy

"B-cellCELL responses in patientsORGANISM who have recovered from severe acute respiratory syndrome target a dominant site in the S2 domain of the surface spike glycoprotein",

displaCy

"Identification of two neutralizing regions on the severe acute respiratory syndrome coronavirusORGANISM spike glycoprotein produced from the mammalian expression system",

displaCy

"Amino acids 1055AMINO_ACID to 1192 in the S2 region of severe acute respiratory syndrome coronavirus S protein induce neutralizing antibodies: implications for the development of vaccines and antiviral agents",

displaCy

A DNACELLULAR_COMPONENT vaccine induces SARS coronavirusORGANISM neutralization and protective immunity in miceORGANISM",

displaCy

"Severe acute respiratory syndrome coronavirusORGANISM spike protein expressed by attenuated vaccinia virusORGANISM protectively immunizes miceORGANISM",

displaCy

"Contributions of the structural proteins of severe acute respiratory syndrome coronavirusORGANISM to protective immunity",

displaCy

"Evaluation of human monoclonal antibodyORGANISM 80R for immunoprophylaxis of severe acute respiratory syndrome by an animal study, epitope mapping, and analysis of spike variants",

displaCy

"Development and characterization of a severe acute respiratory syndrome-associated coronavirus-neutralizing human monoclonal antibodyORGANISM that provides effective immunoprophylaxis in miceORGANISM",

displaCy

"Potent neutralization of severe acute respiratory syndrome (SARS) coronavirusORGANISM by a human mAbORGANISM to S1 protein that blocks receptor association",

displaCy

"Prior infection and passive transfer of neutralizing antibody prevent replication of severe acute respiratory syndrome coronavirusORGANISM in the respiratory tract of miceORGANISM",

displaCy

"Nucleocapsid-independent assembly of coronavirus-like particlesGENE_OR_GENE_PRODUCT by co-expression of viral envelope protein genes",

displaCy

"Genetic analysis of determinants for spike glycoprotein assembly into murine coronavirus virionsORGANISM: distinct roles for charge-richCELL and cysteine-rich regions of the endodomain",

displaCy

"Mapping of the coronavirus membrane protein domainsORGANISM involved in interaction with the spike protein",

displaCy

"Protein interactions during coronavirusORGANISM assembly",

displaCy

"Cooperation of an RNA packaging signal and a viral envelope protein in coronavirusORGANISM RNA packaging",

displaCy

"Genetic evidence for a structural interaction between the carboxy termini of the membraneCELLULAR_COMPONENT and nucleocapsidGENE_OR_GENE_PRODUCT proteins of mouse hepatitis virusORGANISM",

displaCy

"Characterization of the coronavirus MORGANISM protein and nucleocapsidGENE_OR_GENE_PRODUCT interaction in infected cellsCELL",

displaCy

"Isolation of coronavirus envelope glycoproteinsORGANISM and interaction with the viral nucleocapsidORGANISM",

displaCy

"NucleocapsidindependentSIMPLE_CHEMICAL specific viral RNA packaging via viral envelope protein and viral RNA signal",

displaCy

"Release of coronavirus EORGANISM protein in membrane vesiclesCELLULAR_COMPONENT from virus-infected cellsCELL and E proteinexpressing cellsCELL",

displaCy

"Coronavirus pseudoparticlesORGANISM formed with recombinant M and E proteinsGENE_OR_GENE_PRODUCT induce alpha interferonGENE_OR_GENE_PRODUCT synthesis by leukocytesCELL",

displaCy

"Severe acute respiratory syndrome coronavirus 7a accessoryORGANISM protein is a viral structural protein",

displaCy

"Efficient assembly and release of SARS coronavirus-like particlesORGANISM by a heterologous expression system",

displaCy

"ImmuneCELL responses against severe acute respiratory syndrome coronavirusORGANISM induced by virus-like particlesORGANISM in miceORGANISM",

displaCy

"Generation of synthetic severe acute respiratory syndrome coronavirus pseudoparticlesORGANISM: implications for assembly and vaccine production",

displaCy

"Retargeting of coronavirusORGANISM by substitution of the spike glycoprotein ectodomain: crossing the host cellCELL species barrier",

displaCy

"Severe acute respiratory syndrome coronavirusORGANISM infection of miceORGANISM transgenic for the human Angiotensin-converting enzyme 2 virus receptorORGANISM",

displaCy

"Envelope glycoprotein interactions in coronavirusORGANISM assembly",

displaCy

"Immunogenicity of a polyvalent HIV-1ORGANISM candidate vaccine based on fourteen wild type gp120GENE_OR_GENE_PRODUCT proteins in golden hamstersORGANISM",

displaCy

"A pre-S/S CHO-derived hepatitis B virus vaccine protects woodchucksORGANISM from WHVORGANISM productive infection",

displaCy

"DNACELLULAR_COMPONENT immunization with the cysteine-rich interdomain region 1 of the Plasmodium falciparum variant antigen elicits limited cross-reactive antibody responses",

displaCy

"chimeric live, attenuated vaccine (ChimeriVaxSIMPLE_CHEMICAL) incorporating the envelope genes of Japanese encephalitis (SA14-14-2) virusORGANISM and the capsidORGANISM and nonstructuralAMINO_ACID genes of yellow fever (17D) virus is safe, immunogenic and protective in non-human primatesORGANISM",

displaCy

"Induction of protective immunity in animals vaccinated with recombinant vaccinia virusesORGANISM that express PreMGENE_OR_GENE_PRODUCT and E glycoproteinsGENE_OR_GENE_PRODUCT of Japanese encephalitis virus",

displaCy

"Long-term protection from SARS coronavirusORGANISM infection conferred by a single immunization with an attenuated VSV-basedGENE_OR_GENE_PRODUCT vaccine",

displaCy

"Recombinant modified vaccinia virus AnkaraORGANISM expressing the spike glycoprotein of severe acute respiratory syndrome coronavirusORGANISM induces protective neutralizing antibodies primarily targeting the receptor binding region",

displaCy

"A double-inactivated whole virus candidate SARS coronavirus vaccineORGANISM stimulates neutralising and protective antibody responses",

displaCy

"Inactivated SARS-CoV vaccineORGANISM prepared from whole virus induces a high level of neutralizing antibodies in BALB/c miceORGANISM",

displaCy

"Immunogenicity of SARS inactivated vaccine in BALB/c miceORGANISM",

displaCy

"Inactivated SARS-CoV vaccineORGANISM elicits high titers of spike protein-specific antibodies that block receptor binding and virus entry",

displaCy

"SARS coronavirusORGANISM spike polypeptide DNACELLULAR_COMPONENT vaccine priming with recombinant spike polypeptide from Escherichia coliORGANISM as booster induces high titer of neutralizing antibody against SARS coronavirusORGANISM",

displaCy

"Antigenic and immunogenic characterization of recombinant baculovirus-expressed severe acute respiratory syndrome coronavirusORGANISM spike protein: implication for vaccine design",

displaCy

"Evaluation of modified vaccinia virus AnkaraORGANISM based recombinant SARS vaccine in ferretsORGANISM",

displaCy

"Exosomal vaccines containing the S protein of the SARS coronavirusORGANISM induce high levels of neutralizing antibodies",

displaCy

"Mucosal immunization with surface-displayed severe acute respiratory syndrome coronavirusORGANISM spike protein on Lactobacillus caseiSIMPLE_CHEMICAL induces neutralizing antibodies in miceORGANISM",

displaCy

"Switching species tropism: an effective way to manipulate the feline coronavirusORGANISM genome",

displaCy

"Assembly of spikes into coronavirus particlesORGANISM is mediated by the carboxy-terminal domain of the spike protein",

displaCy

"Virus-like particlesORGANISM: passport to immune recognition",

displaCy

"Human monoclonal antibodyORGANISM as prophylaxis for SARS coronavirusORGANISM infection in ferretsORGANISM",

displaCy

"Receptor-binding domain of SARS-CoVORGANISM spike protein induces long-term protective immunity in an animal model",

displaCy

"Recombinant adeno-associated virus expressing the receptor-binding domain of severe acute respiratory syndrome coronavirus SORGANISM protein elicits neutralizing antibodies: implication for developing SARS vaccines",

displaCy

"Neutralizing antibody and protective immunity to SARS coronavirusORGANISM infection of miceORGANISM induced by a soluble recombinant polypeptide containing an N-terminal segment of the spike glycoprotein",

displaCy

"Therapy with a severe acute respiratory syndrome-associated coronavirus-neutralizing human monoclonal antibodyORGANISM reduces disease severity and viral burden in golden Syrian hamstersORGANISM",

displaCy

"VaccineORGANISM efficacy in senescent miceORGANISM challenged with recombinant SARS-CoV bearing epidemic and zoonotic spike variants",

displaCy

"Early death after felineORGANISM infectious peritonitis virus challenge due to recombinant vaccinia virusORGANISM immunization",

displaCy

"Immunopathogenesis of coronavirusORGANISM infections: implications for SARS",

displaCy

"Antibody-mediated enhancement of disease in felineORGANISM infectious peritonitis: comparisons with dengue hemorrhagic fever",

displaCy

"Monoclonal antibodies to the spike protein of feline infectious peritonitis virusORGANISM mediate antibody-dependent enhancement of infection of feline macrophagesORGANISM",

displaCy

"Monoclonal antibody analysis of neutralization and antibody-dependent enhancement of feline infectious peritonitis virusORGANISM",

displaCy

"Evasion of antibody neutralization in emerging severe acute respiratory syndrome coronavirusesORGANISM",

displaCy

"Lethal infection of K18-hACE2 mice infected with severe acute respiratory syndrome coronavirusORGANISM",

displaCy

"Synthetic reconstruction of zoonotic and early humanORGANISM severe acute respiratory syndrome coronavirus isolatesORGANISM that produce fatal disease in aged miceORGANISM",

displaCy

"Severe acute respiratory syndrome coronavirusORGANISM infection of golden Syrian hamstersORGANISM",

displaCy

"Virology: SARS virus infection of catsORGANISM and ferretsORGANISM",

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"CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrixCELLULAR_COMPONENT choice",

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"Characterization of VLPsORGANISM.

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(A) SCoV VLPs, MHV VLPsORGANISM and chimeric VLPsCELL, all of which were produced from 293T cellsCELL, were independently purified by sucroseSIMPLE_CHEMICAL gradient centrifugation and 5 g of purified VLPsCELL were applied to each lane of SDS-PAGE.

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Colloidal Coomassie blue staining (CCB) and Western blot (WB) analysis of purified SCoV VLPsORGANISM, MHV VLPsORGANISM and chimeric VLPsCELL are shown.

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(B) Negative staining of a chimeric VLPCELL.

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"SCoV-specific neutralizing antibody titers of immunized miceORGANISM at 28 days after immunization (A) and SCoVSIMPLE_CHEMICAL titers in lungsORGAN of mice 2ORGANISM days after intranasal challenge with 1 \u00d7 10 6 TCID 50 of SCoVSIMPLE_CHEMICAL at day 28 (B).

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(A) Mean SCoV-specific neutralizing antibody titers of the miceORGANISM at 28 days after i.nORGANISM.

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inoculation of SCoVSIMPLE_CHEMICAL or i.mSIMPLE_CHEMICAL.

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inoculation of placebo (medium from Vero E6 cellsCELL) or 2 g chimeric VLPCELL suspended in PBS or alumSIMPLE_CHEMICAL are shown.

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The lengths of the bars indicate mean virus-specific serumORGANISM neutralizing antibody titers.

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"Figure 3 SCoV-specific neutralizing serumORGANISM_SUBSTANCE antibody titers in the immunized miceORGANISM and SCoVSIMPLE_CHEMICAL titers in lungsORGAN of mice 2ORGANISM days after intranasal challenge with SCoVSIMPLE_CHEMICAL at day 56.

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On day 0, miceORGANISM were either leftORGAN untreated or were inoculated i.nORGANISM.

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with 1 \u00d7 10 6 TCID 50 of SCoVSIMPLE_CHEMICAL or injected i.mSIMPLE_CHEMICAL.

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(A) The graph represents virus-specific serumORGANISM neutralizing antibody titers at 28 days post inoculation.

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The graph represents virus-specificGENE_OR_GENE_PRODUCT, serumneutralizing antibody titers at 56 days after initial inoculation.

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After 2 days, miceORGANISM were euthanized, and the lung virusTISSUE titers were determined.

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The vertical dashed line in (C) denotes the minimal virus detection level in this assay (2.3 log 10 TCID 50 /g lungORGAN).

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"LungORGAN histopathology and immunohistochemistry of the control placebo and immunized miceORGANISM at 2 days post SCoVSIMPLE_CHEMICAL challenge.

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MiceORGANISM (n = 5 for all groups) were inoculated with VeroE6 cellCELL culture fluidsTISSUE twice at days 0 and 28 (A-C), immunized with a mixture of chimeric VLPsCELL and PBS at days 0 and 28 (D and G), chimeric VLPsCELL and alumSIMPLE_CHEMICAL (ESIMPLE_CHEMICAL and HCELL) at days 0 and 28, or live SCoVSIMPLE_CHEMICAL at day 0 only (F and IGENE_OR_GENE_PRODUCT).

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These miceORGANISM were challenged with SCoVSIMPLE_CHEMICAL at day 56 and sacrificed at day 58.

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(A) Bronchiolar epithelial cellsCELL showed swelling and blebbing of the luminal cytoplasmTISSUE, and extensive cellularCELL debris comprised of necrotic epitheliumTISSUE and inflammatory cellsCELL in the airway lumenMULTI-TISSUE_STRUCTURE.

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Moderate peribronchiolar mononuclearCELL inflammatory cell infiltrates are also present (hematoxylin and eosin staining) (magnification, \u00d7200GENE_OR_GENE_PRODUCT).

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(B) Thickening of the bronchiolar interstitial tissuesTISSUE and alveolar wallsTISSUE with mononuclear cellCELL infiltration (magnification, \u00d7200GENE_OR_GENE_PRODUCT).

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(C) SCoV N antigen was distributed in bronchiolar epithelial cellsCELL, as determined by immunohistochemistry (magnification, \u00d7400GENE_OR_GENE_PRODUCT).

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(D-F) Bronchiolar epithelial cells showed rare swelling and blebbing of the luminal cytoplasmMULTI-TISSUE_STRUCTURE, and the rare presence of cellularCELL debris in airwaysTISSUE (magnification, \u00d7200GENE_OR_GENE_PRODUCT).

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The peribronchiolar mononuclearCELL, neutrophilCELL and eosinophil infiltratesTISSUE (D, inset) (magnification, \u00d7400GENE_OR_GENE_PRODUCT).

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(G-I) SCoV antigens were not detected by immunohistochemistry in the lungsORGAN of these miceORGANISM (magnification, \u00d7400GENE_OR_GENE_PRODUCT).

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"Amino acidAMINO_ACID sequences of endodomains of SCoVSIMPLE_CHEMICAL, MHVORGANISM and FIPVORGANISM.

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A CLUSTALW alignmentof the carboxy-terminus of S protein sequences of SCoVSIMPLE_CHEMICAL (strain Urbani, GenBank accession no. AY278741), MHVORGANISM (strain A59, GenBank accession no. NC 001846), and FIPVORGANISM (GenBank accession no. AY994055).

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The transmembraneCELLULAR_COMPONENT domain, cytoplasmicORGANISM_SUBSTANCE domain, cystein-rich region, and charge-richCELLULAR_COMPONENT region are indicated.

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Charge-residues in the membraneCELLULAR_COMPONENT proximal region are shaded.

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"We thank Dr. Vsevolod L. Popov and other members of the ElectronCELLULAR_COMPONENT Microscopy Laboratory and University of Texas Medical Branch for their excellent support for electron microscopy.

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We also thank Susan Baker for anti-MHV serumORGANISM_SUBSTANCE, Robert Atmar for statistics analysis and Bob Couch for valuable suggestions and discussion.

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This work was supported by National Institutes of Health Service Contract AI 65298 and grants AI29984ORGANISM and AI72493.GENE_OR_GENE_PRODUCT

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NI was supported by a fellowship for long-term overseas research for young investigators sponsored by the Ministry of Education, Culture, Sports, Science and TechnologyGENE_OR_GENE_PRODUCT, Japan.